The Impact of substrate texture on cell behavior

Traditionally, cell studies have focused mainly on the effects of biochemical signals on cellular behaviour; however, there is a growing interest in investigating the effects of physical cues on the behaviour of the cells. Development of fabrication techniques has allowed researchers to design topographical cues at the micro and nanometer scale. In this study, we fabricated fibronectin stripes, which mimic some of the frequent patterns that cells are exposed to in their natural microenvironment, and studied the effects of these patterns on 3T3 fibroblasts and human mesenchymal stem cells which are of significant importance in the field of regenerative medicine and tissue engineering. Our results show that cells align and elongate in the direction of the patterns and the morphological features of cell, nucleus and architecture of force-bearing stress fibers of the actin cytoskeleton are all highly dependent on the stripe width. 3D confocal measurements showed that the cell thickness and nuclear shape is regulated through the different arrangement of perinuclear actin stress fibers depending on the stripe sizes. We also observed that when the size of stripes (5 µm) is significantly smaller than the size of cell nuclei (subnuclear stripes), cells exhibit 3 different morphological responses to the patterns. Minimal confinement, branching on the stripes and elongation on a single stripe. The majority of cells and nuclei align themselves with the subnuclear stripes; however, a substantial number of hMSCs nuclei are elongated perpendicular to the direction of the pattern, a finding which we explained using a geometrical model based on cell size. The behaviour of 3T3 cell lines and the hMSCs were compared for the different types of pattern. We designed setups that allow us to perform Raman microspectroscopy both in 2D and 3D topographical patterns in order to be able to study their morpho-chemical effects on cells. We also genetically modified 3T3 fibroblasts to express fluorescent-tagged proteins that visualize the actin cytoskeleton and the nucleus which allow us to perform live cell imaging to study the dynamics of cell behaviour on topographical patterns.Peterhouse College research studentship, Cavendish laboratory research scholarship, Cambridge Trust- IDB 3 years full PhD scholarshi

Microchips and their significance in isolation of circulating tumor cells and monitoring of cancers

In micro-fluid systems, fluids are injected into extremely narrow polymer channels in small amounts such as micro-, nano-, or pico-liter scales. These channels themselves are embedded on tiny chips. Various specialized structures in the chips including pumps, valves, and channels allow the chips to accept different types of fluids to be entered the channel and along with flowing through the channels, exert their effects in the framework of different reactions. The chips are generally crystal, silicon, or elastomer in texture. These highly organized structures are equipped with discharging channels through which products as well as wastes of the reactions are secreted out. A particular advantage regarding the use of fluids in micro-scales over macro-scales lies in the fact that these fluids are much better processed in the chips when they applied as micro-scales. When the laboratory is miniaturized as a microchip and solutions are injected on a micro-scale, this combination makes a specialized construction referred to as "lab-on-chip". Taken together, micro-fluids are among the novel technologies which further than declining the costs; enhancing the test repeatability, sensitivity, accuracy, and speed; are emerged as widespread technology in laboratory diagnosis. They can be utilized for monitoring a wide spectrum of biological disorders including different types of cancers. When these microchips are used for cancer monitoring, circulatory tumor cells play a fundamental role

Extracellular vesicles generated by placental tissues ex vivo: A transport system for immune mediators and growth factors

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144634/1/aji12860_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144634/2/aji12860.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144634/3/aji12860-sup-0001-Supinfo.pd

ナノ及びマイクロパターン化表面による間葉系幹細胞の形態と機能の制御

筑波大学 (University of Tsukuba)201

Strontium substituted hydroxyapatite with β-lactam integrin agonists to enhance mesenchymal cells adhesion and to promote bone regeneration

Multi-functionalization of calcium phosphates to get delivery systems of therapeutic agents is gaining increasing relevance for the development of functional biomaterials aimed to solve problems related to disorders of the muscolo-skeletal system. In this regard, we functionalized Strontium substituted hydroxyapatite (SrHA) with some β-lactam integrin agonists to develop materials with enhanced properties in promoting cell adhesion and activation of intracellular signaling as well as in counteracting abnormal bone resorption. For this purpose, we selected two monocyclic β-lactams on the basis of their activities towards specific integrins on promoting cell adhesion and signalling. The amount of β-lactams loaded on SrHA could be modulated on changing the polarity of the loading solution, from 3.5–24 wt% for compound 1 and from 3.2–8.4 wt% for compound 2. Studies on the release of the β-lactams from the functionalized SrHA in aqueous medium showed an initial burst followed by a steady-release that ensures a small but constant amount of the compounds over time. The new composites were fully characterized. Co-culture of human primary mesenchymal stem cells (hMSC) and human primary osteoclast (OC) demonstrated that the presence of β-lactams on SrHA favors hMSC adhesion and viability, as well as differentiation towards osteoblastic lineage. Moreover, the β-lactams were found to enhance the inhibitory role of Strontium on osteoclast viability and differentiation

Controlling the fate of stem cells through two-and three-dimensional scaffolds based on bioresorbable polymers and graphenen derivatives: a study towards nerve tissue regeneration.

184 p.Neurological disorders are the major cause of long-term impairment and the second largest cause of death worldwide. Hence, there is an urgent need for new treatments that allow the functional recovery of damaged tissue. Among the experimental treatments, bioresorbable polyesters are showing great results in preclinical and clinical trials due to their biocompatibility, tunable degradability, versatility and physicochemical and mechanical properties. In this PhD thesis nanostructured scaffolds based on bioresorbable polymers and graphene oxide were developed to study the attachment, aligned growth and migration of both murine and human stem cells, avoiding the use of extracellular matrix-like compounds coatings. The use of murine neural stem cells allowed to study the differentiation pattern of the cells over the nanostructuredscaffolds, focusing on the achievement of a balanced neuronal and glial support, for a long-term survival of the cultures in vitro. The use of a relatively new source of stem cells, now considered clinical waste, like the dental pulp stem cells, allowed to minimize the ethical concerns and provide an actual alternative for personalized medicine in future therapies. To test this alternative, the regeneration capabilities of the nanostructured scaffolds were studied after the impairment of the rostral migratory stream in a rodent model in vivo. And with the aim of addressing the enhanced restoration capabilities of the personalized advanced medical products combining polymeric materials and human stem cells, the regeneration of the rostral migratory stream was compared when grafting the dental pulp stem cells, alone or in combination with our nanostructured scaffolds.Finally, to better resemble the neural niche in vitro graphene derivatives-based three-dimensional scaffolds with tunable geometry, mechanical and electrical conductive features were fabricated and their effect studied on cell survival and differentiation. Afterward, cerium oxide nanoparticles were incorporated to provide enhanced antioxidant and neuroprotective features and their effect on the establishment of balanced neuronal and glial co-cultures studied.Overall, this thesis gives new insights into the design of polymeric materials based on graphene derivatives for future personalized advanced medical products in combination with human stem cells for the restoration of the nervous syste

Screening Platform for Cell Contact Guidance Based on Inorganic Biomaterial Micro/nanotopographical Gradients

High -throughput screening (HTS) methods based on topography gradients or arrays have been extensively used to investigate cell material interactions. However, it is a huge technological challenge to cost efficiently prepare topographical gradients of inorganic biomaterials due to their inherent material properties. Here, we developed a novel strategy translating PDMS-based wrinkled topography gradients with amplitudes from 49 to 2561 nm and wavelengths between 464 and 7121 nm to inorganic biomaterials (Sio(2), Ti/Tio(2), Cr/Cro(3), and AL(2)O(3)) which are frequently used clinical materials. Optimal substratum conditions promoted human bone-marrow derived mesenchymal stem cell alignment, elongation, cytoskeleton arrangement, filopodia development as well as cell adhesion in vitro, which depended both on topography and interface material. This study displays a positive correlation between cell alignment and the orientation of cytoskeleton, filopodia, and focal adhesions. This platform vastly minimizes the experimental efforts both for inorganic material interface engineering and cell biological assessments in a facile and effective approach. The practical application of the HTS technology is expected to aid in the acceleration of developments of inorganic clinical biomaterials

Grainyhead-like 2 Sensitizes Cells to Natural Killer Cell Cytotoxicity and Promotes the Interferon Response

Our research determined that the epithelial master transcription factor Grainyhead-like 2 (GRHL2) promotes sensitivity to Natural Killer (NK) cell-mediated killing, and modulates the interferon I (IFN-I) response of epithelial cells. Immune surveillance by NK cells constitutes a major selective pressure for circulating tumor cells. Epithelial (GRHL2-expressing) cells exhibited significantly higher rates of NK conjugation, a crucial step in direct cell-mediated cytotoxicity. Mechanistically, GRHL2 upregulates expression of intercellular adhesion molecule 1 (ICAM-1), a cell surface molecule critical for NK to target cell synaptogenesis. GRHL2 epigenetically regulates gene expression, and we found that GRHL2 mutant proteins unable to interact with the epigenetic modifiers p300 or KMT2C/D exhibited altered NK sensitivity, and ICAM-1 expression was lost. In summary, the epithelial state, enforced by GRHL2, kept these cells susceptible to NK-mediated cytolysis. Further, our data suggest GRHL2 critically promotes IFN I production in response to a double strand RNA mimetic. The results from these experiments identify novel roles for GRHL2 in maintaining the relationship between epithelial cells and the innate and adaptive arms of the immune system

Developing sustained dual-drug therapy for tendon sports injuries

Tendon plays an important role in regulating body locomotion and providing additional stability to the body. However, tendon is susceptible to injuries and the healing process could be devastating along with the several issues, namely adhesion formations, slow healing and failure at fixation sites, which have deferred the success of proper tendon healing via tendon tissue engineering. This dissertation thus aims to create a sustained dual-drug therapy to address these issues. For adhesion formation, naproxen sodium (NPS) has been shown to be able to avoid this symptom through inhibiting inflammation process. [Continues.